Supporting document 1 - Food Standards Australia New Zealand

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i



Supporting document

1


Safety assessment


A
pplication

A1085


F
ood

derived from Reduced L
ignin Lucerne

L
ine KK179



Summary and conclusions


Background


A genetically
modified (GM) lucerne line, KK179, has been developed that has reduced
biosynthesis
of guaiacyl lignin (G lignin), a major subunit of lignin. Lignin is a non
-
carbohydrate phenolic polymer deposited in plant cell walls, particularly in the vascular
tissue, and is a contributor to the quality of forage eaten by grazing animals. The Applican
ts
claim that growers will have the option of being able to harvest KK179 several days later than
conventional lucerne without appreciable loss of forage quality

typical in conventional lucerne
at the same growth stage
.


The reduced level of lignin in
lucerne KK179 has been achieved through the introduction of a
partial
caffeoyl CoA 3
-
O
-
methyltransferase

(
CCOMT
) gene sequence

derived from lucerne
(
Medicago sativa
). The gene transcript
acts, via suppression

of the endogenous
CCOMT

gene
,

to reduce the lig
nin level.


It is not intended that KK179 enter the food supply. However, a food approval is sought in
case this inadvertently occurs.


In conducting a safety assessment of food derived from
lucerne line KK179
, a number of
criteria have been addressed
including: a characterisation of the transferred gene
tic material
and

its origin, funct
ion and stability in the lucerne genome
; compositional analyses;
and
evaluation of intended and unin
tended changes.


This safety assessment report addresses only food
safety and nutritional issues

associated
with the GM line
. It therefore does not address:




environmental risks related to the environmental release of GM plants used in food
production



the safety of animal feed or animals fed with feed derived from GM pla
nts



the safety
per se

of food derived from the non
-
GM (conventional) plant.




ii

History of Use


Lucerne is grown primarily for livestock feed and
is grown throughout the world
(approximately 30
million ha) as forage
.

It is often harvested for hay, but can also be made
into silage

and manufactured stock fe
e
d (meal and pellets). The main food products from
M.
sativa

are alfalfa sprouts, comprising sprouted seeds packed into punnets that are used as a
fresh vegetable in
salads, sandwiches, soups and stir
-
fries.
Other alfalfa products are widely
available in specialised stores, for example alfalfa in the form of dried

leaf, health drinks and
teas.


Molecular Characterisation


Explants of
the
lucerne

line

‘R2336’ were transformed via
Agrobacterium
-
mediated
transformation,
the genes of interest being inserted via two separate T
-
DNAs.

T
-
DNA I

contains two
CCOMT

fragments, that when transcribed lead to the production of double
-
stranded RNA
s

(dsRNA
s
) that, via

an
RNA interference (RNAi)

mechanism
,

suppress
endogenous
CCOMT

RNA levels, leading to reduced biosynthesis of G
-
lignin.



In order to select putative transformants, a T
-
DNA II was

also
inserted during the
transformati
on procedure. This contained a

neomycin phosphotransferase II

(
nptII
) coding
region that confers resistance to kanamycin. T
-
DNA II was removed from KK179 by
selection.


Comprehensive

molecular analyses of lucerne line KK179

indicate

there is a single insertion
site at which there is a s
ingle copy of the T
-
DNA I
.
No DNA sequences from T
-
DNA II or from
the backbone of the transformation vector, including antibiotic resistance marker genes, were
transferred to the plant. The introduced genetic elements are stably inherited from one
generati
on to the next.


Northern blot analyses were used to compare the RNA levels associated with the
endogenous
CCOMT

gene in forage and root tissue of KK179.
The data show a clear
reduction in the level of
CCOMT
m
RNA in KK179 compared to the conventional control
and hence that insertion of the
CCOMT

suppression cassette in T
-
DNA I has resulted in
the intended modification.


C
ompositional Analyses


In order
to establish the nutritional adequacy of
forage from lucern
e line KK179,
samples
were analysed for
50 analytes comprising
nutrients; proximates (ash, fat, moisture, and
protein), carbohydrates by calcula
tion, acid detergent fibre
, neutral detergent
fibre
, acid
detergent lignin, minerals,
amino acids
and a number of anti
-
nutrients and secondary
metabolites. In addition,
p
-
coumaric acid, ferulic acid, sinapic acid, total polyphenols and free
phenylalanine were also analysed to evaluate the effect of
CCOMT

suppression on
the
lignin
pathway and cell wall
-
associated metabolites.


As expected, the level
s

of lignin

in general, and G lignin in particular, in KK179 were

statistically
significantly lower than

in the control.
The overall magnitude of the difference
h
owever
was small, and
the
lignin
levels were within the
reference range obtained for non
-
GM reference varieties grown at the same time
. While
the difference in lignin levels between
the GM line and the control is of agronomic significance, in that it enables the forage to be
harvested at
a later date without appreciable loss of forage quality, it is unlikely to have any
nutritional significance to humans given the range of natural variation that exists in lucerne
.


For the remaining analytes,
statistically
significant differences were not
ed in only three
analytes

(
ash, canavanine and ferulic acid
). In all cases the differences

were typically small
iii

and within the reference range obtained for non
-
GM reference varieties grown at the same
time. Any observed differences are therefore considered

to represent the natural variability
that exists within lucerne.



Conclusion


No potential public health and safety concerns have been identified in the assessment of
lucerne line KK179
. On the basis of the data provided in the present Application, and
other
available information, food derived from lucerne line KK179 is considered to be as safe for
human consumption as food derived from conventional lucerne cultivars.





1

TABLE OF CONTENTS


SUMMARY AND CONCLUSI
ONS

................................
................................
.........................

I

LIST OF TABLES

................................
................................
................................
.................

2

LIST OF FIGURES

................................
................................
................................
...............

2

LIST OF
ABBREVIATIONS

................................
................................
................................
..

3

1.

INTRODUCTION

................................
................................
................................
...........

4

2.

HISTORY OF USE

................................
................................
................................
........

4

2.1

H
OST AND

DONOR ORGANISM

................................
................................
......................

4

2.2

O
THER ORGANISMS

................................
................................
................................
.....

6

3.

MOLECULAR CHARACTERI
SATION

................................
................................
..........

6

3.1

M
ETHOD USED IN THE GE
NETIC MODIFICATION

................................
..............................

7

3.2

D
ESCRIPTION OF THE IN
TRODUCED GENETIC MAT
ERIAL

................................
.................

8

3.3

B
REEDING TO OBTAIN LU
CERNE LINE
KK179

................................
...............................

11

3.4

C
HARACTERISATION OF T
HE GENETIC MATERIAL
IN THE PL
ANT

................................
.....

13

3.5

S
TABILITY OF THE GENE
TIC CHANGE

................................
................................
...........

15

3.6

A
NTIBIOTIC RESISTANCE

MARKER GENES

................................
................................
....

16

3.7

C
ONCLUSION

................................
................................
................................
............

16

4.

CHARACTERISATION OF
NOVEL SUBSTANCES

................................
...................

16

4.1

P
OTENTIAL ALLERGENICI
TY
/
TOXICITY OF ANY NOVE
L
ORF
S CREATED BY THE
TRANSFORMATION PROCE
DURE

................................
................................
..................

17

4.2

T
HE EXPRESSION OF
CCOMT

IN
KK179

................................
................................
.....

15

5.

COMPOSITIONAL ANALYS
IS

................................
................................
...................

18

5.1

K
EY COMPONENTS OF LUC
ERNE

................................
................................
.................

18

5.2

S
TUDY DESIGN
,

CONDUCT AND ANALYSIS

................................
................................
....

19

5.3

F
ORAGE COMPOSITION

................................
................................
..............................

20

5.4

C
ONCLUSION

................................
................................
................................
............

25

7.

NUTRITIONAL IMPACT

................................
................................
.............................

26

REFERENCES

................................
................................
................................
...................

26



2

LIST OF
TABLES


Table 1: Importation (kg) to A) Australia, between 2008


2012, and B) New Zealand in 2005
of lucerne seed for sowing/sprouting (by country of origin)

................................
......

5

Table 2: Description of the genetic elements contained in the two T
-
DNAs of PV
-
MSPQ12633

................................
................................
................................
............

8

Table 3: KK179 generations used for various analyses

................................
.......................

12

Table 4: Mean percentage
± S.E.

of proximates and fibre in forage from C
0

Syn1 and KK179

................................
................................
................................
..............................

21

Table
5: Summary of forage lignin unit content in C
0

Syn1 and KK179

...............................

22

Table 6: Mean percentage dry weight (dw)
± S.E.
, relative to tot
al dry weight, of amino acids
in forage from C
0

Syn1 and KK179

................................
................................
........

22

Table 7: Mean values ± S.E. for mineral levels in forage from C0

Syn1 and KK179

............

23

Table 8: Mean levels ± S.E. of other analytes considered in forage from C0 Syn1 and KK179

................................
................................
................................
..............................

24

Table 9: Summary of analyte means found in forage from KK179 that are significantly
( P<0.05) different from those found in forage of t
he control line C
0

Syn1

..............

25



LIST OF
FIGURES


Figure 1: Vector map of plasmid PV
-
MSPQ12633

................................
................................

7

Figure 2: Simplified diagram of the lignin biosynthetic pathways indicating where the CCOMT
enzyme acts and where it would be bl
ocked in KK179

................................
..........

10

Figure 3: Breeding strategy for plants containing event KK179

................................
...........

12

Figure 4: Schematic location and predicted sizes of the five PCR products amplified from
KK179

................................
................................
................................
...................

14




3

L
IST OF ABBREVIATIONS

ADF

acid detergent fibre

ADL

Acid detergent lignin

AOAC

Association of Analytical Communities

BLOSUM

Blocks Substitution Matrix

bp

base pairs

bw

body weight

CaMV

Cauliflower mosaic virus

CCOMT

caffeoyl CoA 3
-
O
-
methyltransferase

DNA

deoxyribonucleic
acid

T
-
DNA

transferred DNA

FARRP

Food Allergy Research and Resource Program

FASTA

Fast Alignment Search Tool
-

All

FD4

Fall dormancy 4

FSANZ

Food Standards Australia New Zealand

fw

fresh weight

G lignin

guaiacyl lignin

GM

genetically modified

ha

hectare

LOQ

limit of quantitation

M
BC

modified backcross

NCBI

National Center for Biotechnology Information

NDF

neutral detergent fibre

nos

nopaline synthase

OECD

Organisation for Economic Co
-
operation and Development

OGTR

Office of the Gene
Technology Regulator

ORF

open reading frame

PAL

phenylalanine ammonia lyase

PCR

polymerase chain reaction

RISC

RNA
-
induced silencing complex

RNA

Ribonucleic acid

RNAi

RNA interference

dsRNA

d
ouble stranded RNA

Poly A
+

RNA

polyadenylated mRNA

S.E.

standard error

Ti

tumour
-
inducing

U.S.

United States of America




4

1.

I
ntroduction


A g
enetically modified (GM)
lucerne

line

with
OECD Unique

Identifier
MON
-
00179
-
5
,
hereaf
ter referred to as lucerne KK179
,
has been developed that
has reduced biosynthesis
of guaiacyl lignin (G lignin), a major subunit of lignin.

Lignin is
a non
-
carbohydrate phenolic
polymer
deposited in plant cell walls, particularly in the vascular tissue
,

and is a contributor
to the quality of forage eaten by
grazing animals. Quality

decr
eases
as the proportion of cell
wall components (cellulose, hemicellulose and lignin) increases.

Total lignin levels in KK179
forage are generally similar to lignin levels in conventional lucerne forage harvested several
days earlier under similar producti
on conditions.
The Applicants claim that growers will have
the option of being able to harvest KK179 several days later than conventional lucerne,
without appreciable loss of forage quality

typical in conventional lucerne at the same growth
stage
.


The red
uced level of lignin in
lucerne KK179 has been achieved through the introduction of a
partial
caffeoyl CoA 3
-
O
-
methyltransferase

(
CCOMT
)
gene sequence (fragment)

derived
from lucerne (
Medicago sativa
). The gene transcript has an inverted repeat and
produces
double
-
stranded ribonucleic acid (dsRNA)

which, via an RNA interference (RNAi) pathway,
suppresses endogenous
CCOMT

RNA levels and results in the reduced biosynthesis of

G lignin. This, in turn, reduces the accumulation of total lignin.


The

Applicant has advised

that lucerne KK179 will

be grown
and used
primarily
in

northern
America and there is no

intention to grow the plant line in Australia or New Zealand.

The
Appl
icant has

anticipated that KK17
9 would be stacked with two Roundup Ready™

lucerne
lines
,

J101 and J163 (OECD Unique Identifiers
MON
-
00101
-
8 and

MON00163
-
7
respectively)
,

the

food from which has been appro
ved by FSANZ
(FSANZ, 2007)
.


It is not intended that KK179 enter the food supply. However, a food approval is sought in
case this inadvertently occurs. In Australia and New Ze
aland, lucerne that is used for human
food is
often
referred to as alfalfa.
Alfalfa would be expected to be consumed in minor
quantities and on an occasional basis.


2.

H
istory of use


2.1

Host

and donor
o
rganism


The host organ
ism is a conventional lucerne

(
Medicago sativa

L. ssp.
sativa
),

belonging to
the family Leguminosae

(Small, 2011)
. The comme
rcial
cultivar ‘R2336
’ was used as the
parental variety for the genetic modi
fication described in this application
.
‘R2336’ is a
proprietary cloned line developed by Forage Genetics International; it was selected for
regenerability from an elite, high
-
yielding, fall
-
dormant breeding population. During
development of KK179, ‘R2336’

transformants were crossed with a non
-
GM male sterile line
designated ‘
Ms208’ (see
Section 3.3
) before

final selection of KK179 from the resulting
progeny. Therefore the cross between ‘R2336’ and ‘Ms208’

(designated as C
0
)
is regarded
as the near
-
isogenic

line for the purposes of comparati
ve
assessment with lucerne KK179


Lucerne

is grown
primarily
for livestock feed

but also has a minor place in the food supply

(OECD, 2005; Bouton, 2012)
.
With respect to feed,
it is grown throughout the world
(approximately 30
million ha) as

forage
1

and is often harvested fo
r
hay, but can also be





1

The term ‘forage’ has a number of definitions but is used in this document to refer to the above
-
ground parts of
rooted plants that would be consumed in the field by livestock
(Small, 2011)
. It can be used as a more general
term that also

encompasses ‘fodder’ which is defined as feed that is first harvested by humans before
being fed to animals.

5

made into silage

and manufactured stock fe
e
d

(
meal and pellets
)
.

The major lucerne
-
producing regions are North America with 11.9 million ha (41%), Europe with 7.12 million ha
(25%), South America with 7 million ha (23%), Asia 2.23 mill
ion ha (8%), Africa (2%) and
Oceania (1%). The leading count
ries in terms of area of lucerne

production (in million ha) are
the US (9), Argentina (6.9), Canada (2), Russia (1.8), Italy (1.3) and China (1.3)
(Yuea
go
and Cash, 2009)
.


Lucerne was
first
introduced into Australia in the early 19
th

century
. A second wave of
introductions occurred in the 1980s following devastation of the crop by exotic insect pests.

Currently Australia has about 3.5 million ha of lucerne under crop

in both irrigated and
dryland situations in all states,

and produces around one million

tonnes of hay
(DPIPWE
Tas, 2011)
.


The main

food product

from
M. sativa

is

alfalfa sprouts, comprising sprouted seeds packed
into
punnets that

are used as a fresh vegetable in salads, sandwiches, soups and stir
-
fries.
Consumption would be expected to be in minor quantities on an occasional basis
(OECD,
2005)
.
Alfalfa sprouts are not permitted to be imported to Australia

or New Zealand
. Within
Australia, the seed used for making sprout
s is larg
ely grown in Australia

and accounts for
approximately 4% of total lucerne seed production
(FSANZ, 2011)
. I
t is possible a proportion
of imported seed may
also
be
used for sprouts but s
ince there is no discrimination in end
use of seed imported as ‘seed for
sowing’
(Table 1
A
)
it is difficult to know how much
is used
for pasture and how much may

be used by

the sprout industry
.

In New Zealand,
production
of lucerne for livestock and alfalfa sprouts relies on seed imported from large breeding
programmes in the US, Australia and Europe (Table 1B); approximately

20
% of this imported
seed is used for sprouting.


Table
1
: Importation (kg) to
A)
Australia, between 2008


2012,
and B) New Zealand in 2005
of lucerne seed for sowing
/sprouting

(by country of origin)

A)

Country of origin

2008

2009

2010

2011

2012

Australia (Re
-
imports)

37,500

58,711

73,322



11,750

United States of America

19,594

2,214

1,615

6,260

4,406

New Zealand





200



200

Netherlands







250



Total

57,094

60,925

75,137

6,510

16,356

Source: ABS imported food data


















B)

6


Source: Ministry for Primary Industries
-

http://www.biosecurity.govt.nz/related/related_faqs/ihs/search?page=2&expand=2475


Alfalfa products in the form of dried leaf,
protein supplement,
tablets, capsules, extracts,
health drinks, tonics and teas are widely available in specialised stores

(Bora and Sharma,
2011; Mielmann, 2013)
.

Some of these products may be regulated as foods (e.g. dried leaf,
health drinks

and teas), while other products would be regulated as
therapeutic goods and
dietary supplements (e.g. protein supplements, tablets, capsules and extracts).
Another
possible food product that

can be derived from the lucerne

plant is bee pollen

(Krell, 1996)
.


2.2

Other organisms


Genetic elements from several other organisms have been used in the genet
ic

modification
of lucerne KK179

(refer to

Table 2
). These non
-
coding sequences are used to drive,
enhance or

terminate expression of the inserted DNA fragment
. None of the sources of
these genetic elements is associated with toxic or allergenic responses in humans. The
genetic eleme
nts derived from

the

plant pathogen

Agrobacterium tumefaciens

are not
pathogenic
in themselves

and do not cause path
og
enic symptoms in lucerne KK179
.


It is noted that an antibiotic resistance gene
(
nptII
)
derived from
E. coli

was also used in the
initial transformation but that this gene (and its regulatory elements) was segregated out
during subsequent selection of line KK179 (see discussion in
Section 3.1
).


3.

M
olecular characterisation


Molecular characterisation is necessary

to provide an understanding of the genetic material
introduced into the host genome and helps to frame the subsequent parts of the safety
assessment. The molecular characterisation
addresses three main aspects:



the transformation method together with a de
tailed description of the DNA sequences
introduced to the host genome




a

characterisation of the inserted DNA including any rearrangements that may have
occurred as a consequence of the transformation



the genetic stability of the inserted DNA and any accompanying expressed traits.



Studies

submitted:


2011.

Molecular Characterization of Reduced Lignin Alfalfa KK179.
MSL0023299.

Monsanto
Company

(unpublished)
.

2011. Stability of the DNA Insert in KK179

Across Multiple

Generations.
MSL0023312.
Monsanto
Company (unpublished)

2012. Bioinformatics Evaluation of the Transfer DNA Insert

in KK179 Utilizing the AD_2012,
TOX_2012 and PRT_2012 Databases.

MSL0024048
. Monsanto Company (unpublished)

2012. Bioinforma
tics Evaluation of DNA Sequences

Flanking the 5' and 3' Junctions of Inserted DNA
in KK179: Assessment of

Putative Polypeptides.
MSL0023975
. Monsanto Company (unpublished)

7

2011. Analysis of the Endogenous CCOMT RNA Level in Alfalfa KK179.
MSL0023329
.
Monsanto
Company (unpublished).

2011. Heritability of the KK179 Insert in the MBC2, MBC3,

and Syn1 Populations.
RPN
-
2010
-
0705
.
Monsanto Company (unpublished)

2011. Lignin Analysis of Forage fro
m Multiple Generations of KK179
Alfalfa.
RAR
-
2011
-
0129
.
Monsant
o Company (unpublished).


3.1

Method used in the genetic modification


E
xplants of lucerne ‘R2336’ were

transformed via
Agrobacterium
-
mediated transformation.
The genes of interest were
inserted via two
separate T
-
DNAs (each with

their own Right and
Left Border) into plasmid PV
-
MSPQ12633
(refer
to

Figure 1
)
.
The

border sequences were
isolated from the tumour
-
inducing (Ti
)
plasmid of
Agrobacterium tumefaciens

and normally
delimit the DNA sequence (T
-
DNA) transferred into the plant
(Zambr
yski, 1988)
.

During the
transformation procedure a portion of each border is expected to be integrated into the host
genome
(Tzfira
et al
., 2004)
.


Basically,
leaf explants

were co
-
cultivated with the
Agrobacterium tumefaciens

containing
the binary v
ector
PV
-
MSPQ12633
.

Putative transformants

were selected on a medium
containing
the antibiotics kanamycin and timentin and

s
urviving
leaf pieces were then
regenerated via somatic embryogenesis
(Schenk and Hildebrand
t, 1972; Walker and Sato,
1981)
.

Rooted plants (T
0
) were
transferred to
the gr
eenhouse for growth and further
assessment.


The T
0

plants were crossed to a non
-
GM male sterile line (Ms208) to produce F
1

plants in
which the unlinked insertions of T
-
DNA I and T
-
DNA II were segregated. Subsequent to this,
plants that were positive for T
-
DNA I (containing the
CCOMT

fragment) and negative for T
-
DNA II (containing the antibiotic resistance gene
nptII
) were identified by polymerase chain
reaction (PCR).

KK
179, an individual F
1

plant, was selected as the lead event (P
0
) based
on
its superior characteristics and absence of T
-
DNA II.



Figure
1
: Vector map of plasmid
PV
-
MSPQ12633

8

3.2

Description

of
the
introduced

gene
tic material


Information on the genetic elements in the
two
T
-
DNA insert
s

is summarised
in

Table 2
.


Table
2
: Description of the genetic elements contained in the
two
T
-
DNA
s

of
PV
-
MSPQ12633

Genetic
element

bp location
on
pSYN15954

Size
(bp)

Source

Orient.

Description & Function

References

T
-
DNA I

Left Border

1

-

442

442

Agrobacterium
tumefaciens

Anti
-
clockwise



Required for the transfer of the
T
-
DNA into the plant cell

Barker
et al
(1983)
;
Zambryski
et
al.
(1982)

Intervening
sequence

443
-

490

48





Cloning sequence


Pal2
promoter

491
-

1567

1077

Paseolus
vulgaris

(bean)

Clockwise



D
rives transcription

of the
COMMT
fragment within
vascular tissue and thus allows it
to
mirror

the pattern of lignin
deposition.

Cramer
et al
.
(1462/id/d}

Intervening
sequence

1568
-

1584

17



Cloning sequence


CCOMT

1585
-

2103

519

Medicago

sativa

(lucerne)

Anti
-
c
lockwise



Partial
c
oding sequence of the

CCOMT
gene



Together with the inverted
(clockwise) repeat, s
uppresses
expression of the endogenous
CCOMT

gene

Inoue
et al

(1998)

Intervening
sequence

2104
-

2110

7





Cloning sequence



CCOMT

2111
-

2410

300

Medicago
sativa
(lucerne)

Clockwise



Partial coding sequence of the
CCOMT
gene



Together with the inverted (anti
-
clockwise)
repeat, s
uppresses
expression of the endogenous
CCOMT

gene

Inoue
et al

(1998)

Intervening
sequence

2411
-

2418

6





Cloning sequence


nos

terminator

2419
-

2671

253

Agrobacterium
tumefaciens

Clockwise



3 UTR sequence of the
nopaline
synthase

(nos)

gene



Transcriptional terminator

Depicket
et al.

(1982)
; Fraley
et al.
(1983)

Intervening
sequence

2672
-

2727

56





Cloning sequence


Right
Border

2728
-

3084

357

Agrobacterium
tumefaciens

Anti
-
clockwise



Required for the transfer of the
T
-
DNA into the plant cell

Zambryski
et
al.
(1982)

Vector Backbone (3951

bp)

T
-
DNA II

Left Border

7036
-

7477

442

Agrobacterium
tumefaciens

Anti
-
clockwise



Required for the transfer of the
T
-
DNA into the plant cell

Barker
et al
(1983)
;
Zambryski
et
al.
(1982)

Intervening
sequence

7478
-

7527

50





Cloning sequence


35S
promoter

7528
-

7851

324

Cauliflower
mosaic virus

Clockwise



Drives constitutive expression of
the
nptII

gene


Odell
et al.

(1985)

Intervening
sequence

7852
-

7884

33





Cloning sequence


nptII

7885
-

8679

795

Esherichia coli

Clockwise



Coding sequence of the
neo
gene from transposon Tn
5,
encoding neomycin transferase
II



Confers
resistance to neomycin
and kanamycin


for selection
purposes
.

Fraley
et al.

(1983)

9

Genetic
element

bp location
on
pSYN15954

Size
(bp)

Source

Orient.

Description & Function

References

Intervening
sequence

8680
-

8710

31





Cloning sequence


nos

terminator

3663
-

3915

253

Agrobacterium
tumefaciens

Clockwise



3 UTR sequence of the
nopaline
synthase

(nos)

gene



Transcriptional terminator

Depicket
et al.

(1982)
; Fraley
et al.

(1983)

Intervening
sequence

8964
-

9048

8
5





Cloning sequence



Right

Border

9049
-

9405

357

Agrobacterium
tumefaciens

Anti
-
clockwise



Required for the transfer of the
T
-
DNA into the plant cell

Zambryski
et
al.
(1982)

Vector Backbone (1,203 bp)






3.2.1

T
-
DNA I


Background on lignin biosynthesis


Lignin is a feature of plants adapted to a terrestrial way of life
(Vanholme et al., 2010; Weng
and Chapple, 2010)
. It

is a complex cross
-
linked polymer that is deposited in plant
secondary walls (i.e. walls laid down in cells that have stopped expanding and started
differentiating);

its function is to cement together and anchor the cellulose fibres in the wall
and hence to provide structural support, especially to the vascular system

and aerial parts
.
After cellulose, lignins are the most abundant organic
polymers

known
(Buchanan
et al
.,
2000)
.


The main building blocks
of lignin
are

the hydroxycinnamyl alcohols (monolignols) of which

there are three main types


guaiacyl (G), syringyl (S) and
p
-
hydroxyphenyl (H).
G lignin is
produced predominantly via caffeoyl CoA.
The
CCOMT

gene encodes an enzyme
(CCOMT)
that methylates caffeoyl Co
A

to

produce feruloyl CoA in the pathway th
at leads to the
production of G
lignin monomers
(Guo
et al
., 2001; Vanholme
et al
., 2010; Zhou
et al
., 2011)

(
see Figure 2
).
G
lignin occurs in all
vascular
plants (i.e. angiosperms, gymnosperms
, ferns,
lycophytes).
I
n lucerne, G and S

lignin

make up approxi
mately 95% of the total lignin.


Figure 2 shows that CCOMT is also normally required for the production of S lignin.
However
, if CCOMT is knocked out

or reduced

(as would occur in KK179), then another
enzyme, cinnamoyl CoA reductase 2 (CCR2) is upregulated and
allows continued synthesis
of S lignin
(Zhou
et al
., 2011)
.


10



Figure
2
: Simplified diagram of the lignin biosynthetic pathways indicating where the CCOMT
enzyme acts and where it
would be blocked in KK179
. Major pathway for each
monolignol is represented by large coloured arrows
.
A
dapted from Sticklen
(2008)
.


The CCOMT expression
cassette


The
suppression cassette
in lucerne KK179
contains two
CCOMT

partial
fragments, one

in
the clockwise orientation and the other in the anti
-
clockwise (inverted) orientation. The
fragment
was isolat
ed and cloned from lucerne.
When transcribed

the two fragments
, via an
inverted
repeat,

lead to the production of double
-
stranded RNA (dsRNA)
in the form of a
structure known as a hairpin
that, via RNA interference (RNAi)
,

suppresses endogenous
CCOMT

RNA levels, leading to the reduced biosynthesis o
f G
-
lignin
(see e.g. Guo
et al
.,
2001)
.

RNAi is a
naturally
-
occurring
RNA
-
based
mechanism that
is used by
eukaryotes,
including plants,
to modulate
endogenous
gene expression

as well as destroy foreign RNA
including viral RNA

(Parrott
et al
., 2010)
.
In plants RNAi plays a
fundamental
role in all
aspects of growth and
development
(Bonnet
et al
., 2006)
.


In RNAi t
he dsRNA
hairpin
that is formed

is cleaved into small
ds
RNAs
,

approximately 21
-

24

nucleotides long,
via an endogenously occurring protein known as Dicer

(Hammond,
2005)
.
These mature small dsRNA duplexes contain an interfering antisense strand (the
guide strand)
,

which is complementary to the target mRNA sequence
,

and a passenger
strand. The

guide strand is incorporated i
nto
a multiprotein complex known as the RNA
-
i
nduced silencing complex (RISC)

leading
,

in the case of KK179,
to the targeted destruction
of the
mRNA transcribed from the endogenous
CCOMT

gene
. The result is that production
of CCOMT is suppressed
.


The
CCOMT

coding region in

T
-
DNA

I

is

driven by
a

phenylalanine
ammonia lyase

(
Pal2
)
promoter from
the common bean (
Phaseolus vulgaris
)
. PAL is a key regulatory enzyme in
plant metabolism and is particularly associated with lignin biosynthesis
(Cramer
et al
., 1989)
.
Therefore use of the
Pal2

promoter ensures transcription of the
CCOMT

fragment in a
pattern of expression that is similar to the pattern of lignin deposition within vascular tissue.
The coding region is terminated by a sequence from the 3’ end of the
nopaline synthase
(nos)

gene from
the soil bacterium
Agrobacterium tumefaciens.





11

3.2.2

T
-
DNA II


In order to be able to select putative transformants, a second T
-
DNA was inserted during the
transformation procedure. This contained the
neomycin phosphotransferase II

(
nptII
) coding
region derived from transposon Tn5 of the bacterium
Escherichia coli
.
Th
e gene confers
resistance to kanamycin. It was under the regulation of the constitutive Cauliflower mosaic
virus (CaMV) 35S promoter

and the
nos

terminator. As discussed in
Section 3.1, T
-
DNA II
was removed from KK179 by selection.


3.3

Breeding to obtain
lucerne

line
KK179


A breeding programme was undertaken for the purposes of:



obtaining generations suitable for analysing the molecular and genetic ch
aracteristics
of lucerne KK179



ensuring that the
KK179

event is incorporated into elite
proprietary
breeding line(s)
for commercialisation
.
.


The breeding pedigree for the various generations is given
in

Figure 3
.


I
nitial transformants (
T
0

plants
)

were crossed to a non
-
GM male sterile line (Ms208) to
produce F
1

plants. Following selection for plants with desirable characteristics and not
containing T
-
DNA II, a single plant (P
0
) designated KK179 was obtained
. Lucerne is a
n
autoploid,

self
-
incompatible outcrossing species
which

cannot be self
-
pollinated

to produce

pure isogenic lines because of severe inbreeding depression

(see discussion and references
in Katepa
-
Mupondwa
et al
., 2002)
.

Subsequent generations were the
refore developed by
traditional lucerne breeding techniques. The P
0

plant was hand
-
crossed with each of 10 elite
lucerne genotypes with a fall dormancy
4 (FD4
)
2

phenotype. T
he FD4 plants were used as
the female seed parents and this breeding step is known
as a modified backcross (MBC
).
The resulting progeny are designated MBC1 and were hand crossed with the same 10 elite

FD4 genotypes to produce MBC2. Eighty MBC2 plants

shown to be positive (by
endpoint
TaqMan PCR) for the
CCOMT

suppression cassette

were th
en crossed amongst themselves
in a breeding step known as a polycross to produce the Syn1 generation, the KK179
preferred population for entry into commercial variety development.

Pollen from 20 MBC2
plants shown to be positive for the
CCOMT

suppression ca
ssette was used to pollinate an
FD4 population to produce MBC3 seed
.
Syn1 Adv was a subsequent synthetic population
produced by crossing the Syn1 population in a polycross.


An identical breedin
g process was followed using a

C
0

(the cross between ‘R2336’
and
‘Ms208’)

plant (see Section 2.1) instead

of the T
0

plants in order to produce populations that
could be used as conventional comparators.






2

Fall dormancy refers to the adaptation of lucerne to environments, including shortening photoperiods
and declining temperatures in late summer and autumn. FD is usually divided into three types:
dormant (FD 1
-

3 classes), semi or intermediate dormant (FD
4
-
6 classes), and non
-
dormant (FD 7
-

9 classes). In autumn, dormant varieties grow very slowly or cease to grow and favour the synthesis
and accumulation of soluble sugars, enabling the crops to survive over a hard winter
(Small, 2011)

12


Figure
3
: Breeding strategy for plants containing event
KK179




Table 3

indicates

the generations that were used in the various stu
dies character
ising
lucerne KK179.


Table
3
: KK179

generations used for various analyses

Analysis

KK179

Generation used

Control
(s)

used

Molecular characterisation

P
0

R2336; Ms208;
C
0

Mendelian inheritance

MBC2, MBC3, Syn1


Genetic stability

P
o
; MBC1; MBC2; Syn1


Phenotypic stability

MBC1, Syn1, Syn1 Adv

C
0
-
derived
equivalents of the
3 generations

Northern blot analysis

Syn1

C
0

Syn1

Compositional analyses

Sy
n1

C
0

Syn1




13

3.4

Characterisation of the genetic material

in the plant


A range of analyses
was
undertaken to characterise the genetic modification
in
lucerne

line
KK179
.
These
i
n
cluded
:

DNA sequence,
determination of
insert copy number and integrity;
Open Reading
Frame (
ORF
)

analysis

of inserted DNA

as well as flanking and junction
regions

and Northern blot analysis to determine
whether the level of transcription of the
endogenous
CCOMT

gene had been down
-
regulated as predicted.


3.4.1

Insert characterisation


Genom
ic DNA was obtained from verified
leaf tissue of
P
o

generation lucerne KK179

and
analysed using Southern blotting to determine co
py number, insertion site(s)
,
presence/absence of plasmid backbone and

presence/absence of T
-
DNA II. PCR and DNA
sequence analy
sis were used to provide the DNA sequence of the insert and flanking
regions and to demonstrate the in
tactness

and organisation of the insertion site.


Transgene copy number, insertion site, T
-
DNA II presence/absence and plasmid backbone
analysis


Copy
number, and insertion site of T
-
DNA I were evaluated by digesting the DNA from P
0

with two sets of restriction enzymes designed to cleave once within the inserted DNA and
once within each flanking region. If T
-
DNA I sequences are present as one copy at a s
ingle
integration site in KK179, then a specific banding pattern would be predicted. The resulting
DNA fragments were separated and transferred to a membrane for sequential hybridisation
with five radiolabelled
probes (see red lines labelled 1


4 in the c
entre of Figure 1) that
represented functional elements within T
-
DNA I.


The presence/absence of T
-
DNA II was assessed by using a single T
-
DNA II probe (see
blue line labelled 5 in Figure 1). This probe contained sequences homologous to the nos 3’
UTR seq
uence present in the T
-
DNA I. Thus, the presence of T
-
DNA II sequences would be
indicated by the appearance of hybridization bands in the Southern blot additional to the
band generated by the T
-
DNA I homology.


The presence/absence of plasmid backbone was
assessed by using four backbone probes
(see green lines in the centre of Figure 1). The presence of backbone sequences would be
indicated by the appearance of hybridization bands

on the Southern blot.


A positive control (DNA from ‘C
0
’ spiked with either d
igested PV
-
MSPQ12633 DNA and/or
probe template(s))

was also

included in the Southern blot analyses
.

C
0

DNA digested with
appropriate restriction enzymes was
always used as a negative control and R2336
-

and
Ms208
-
digested DNA were used in
blots using Probe 3. Probe 3 covers the
CCOMT

region
of PV
-
MSPQ12633 which therefore contains sequences identical to the endogenous
CCOMT

present in the lucerne genome. Thus, it would be expected that the random
segregation of endogenous
CCOMT

in d
ifferent

non
-
GM lucerne gen
omes would lead to
different banding patterns when probed with Probe 3. The KK179 event was generated by
crossing transformed ‘R2336’ with non
-
GM ‘Ms208’ and in order to show all endogenous
CCOMT

alleles, both non
-
GM parents were include
d in addition to C
0

as negative controls.

A
hybridisation band that appeared in KK179 as well as in one or both of ‘R2336’ and ‘Ms208’
would indicate endogenous
CCOMT

not associated with the transformation event.


The Southern blot analyses indicated that
there is a single insert of the
CCOMT

suppression
cassette in event KK179 and that the arrangement of the genetic material is the same as
that in the T
-
DNA I of the PV
-
MSPQ12633
plasmid (refer to Figure 1
). No T
-
DNA II or
plasmid backbone sequences are
present in KK179.


14

Sequence analysis


Five

overlapping PCR fragments

spanning the
insert
and adjacent flanking DNA sequences
in event KK179
(
Figure 4
)
were amplified

and purified before being sequenced using BigDye
terminator chemistry (Applied Biosystems, Foster City, CA).

T
he sequences were
then
aligned
to obtain a consensus sequence which was compared to the PV
-
MSPQ12633
sequence.

As a control, PCR using the same t
en pairs of primers was also performed on
genomic DNA from ‘R2336’.



Figure
4
: Schematic location
and predicted sizes
of the five

PCR products

amplified
from KK179

As expected, no PCR products were obtained for ‘R2336’ DNA. The reactions using KK179
DNA produced band sizes as
predicted
,

thereby

confirming the organisation of the insert
was the same as

that for T
-
DNA I.

The sequencing analysis showed that the insert i
s 2,582
bp in length and aligns to the T
-
DNA I sequence in PV
-
MSPQ12633 beginning at base 168
in the Left Border region and ending at base 2,749 in the Right Border
region (
refer to Table
2
).


Analysis of the flankin
g regions provided sequences of

1047 bp
at the 5’ end and 1256 bp at
the 3’ end.


Integrity of insertion site


PCR and sequence analyses
were performed on genomic DNA from KK179 (P
0
) and
‘R2336’. Two primers were use
d



a forward primer specific to the 5’ flanking region of the
insert and a reverse primer specific to the 3’ flanking region.
PCR of both ‘R2336’ and
KK179 generated a PCR product of approx.. 0.7 kb. The product from ‘R2336’ was
sequenced and then a

consen
sus sequence was generated, by compiling multiple
sequencing reactions performed on the verified PCR product, and then aligned to the 5’ and
3’ flanking sequences of the KK179 insert.


The alignment showed that base 742 to base 1047 of the 5’ flanking regi
on of the KK179
insert are identical
with

base 1 to base 306 of ‘R2336’ except for one base, and that base
3630 to base 3855 of the KK179 insert are identical
with

base 409 to base 634 of ‘R2336’
except for one base. This suggest
s

that 102 bases (between base 307


base 408)
of the
‘R2336’ genome
were deleted as a result of

the T
-
DNA integration
.


3.4.2

Novel o
pen reading
frame (ORF) analysis


An

in silico

analysis of the flanking regions was

done to determine whether any novel ORFs
had been created in KK179. Each analysis comprised a search of six
-
frame translations
between stop codons

(TGA, TAG, TAA) for sequences coding for eight amino acids or
greater.

Five

ORF
s
in the 5’ flanking region a
nd five in the 3’ flanking region were identified
.
A discussion of the
bioinfo
rmatic analysis of these
ORFs

is given
in Section 4.1.


15

For the DNA in the insert, the DNA sequences in the sense and anti
-
sense strands were
translated to yield 6 reading frames

and all sequences were then translated using DNAStar,
EditSeq (Version 8.0.2). The resultant amino acid sequences were used for bioinformatic
analyses described
in Section 4.1.


3.5

The expression of
endogenous
CCOMT

in KK179


Norther
n blot analyses were used to compare the RNA levels associated with the
endogenous
CCOMT

gene in forage and root tissue of KK179.

Since the intention of the
genetic modification is to reduce expression of this gene by RNAi, it would be expected that
the
CC
OMT

mRNA levels would be reduced in KK179.


Four replicates of forage and root tissue from verified plants
(Syn1 generation) of KK179 and
C
0

Syn1

were harvested and total RNA was extracted. From this
, PolyA
+

RNA (i.
e.
polyadenylated mRNA) was extracted and

run on Northern blots probed with a radiolabelled
CCOMT

probe amplified from C
0

genomic DNA. An actin probe was also used for the
purpose of demonstrating the quality and relative amount of each PolyA
+

sample.

PolyA
+
RNA from the forage tissue of the conventional control produced a strong
hybridization signal at the expected molecular weight of ~1.1 kb for the
CCOMT
transcript, whereas no detectable hybridi
s
ation signal was produced from the polyA
+
RNA isolated from th
e forage tissue of KK179. Similar results were obtained for the
PolyA
+
RNA from root tissue except that the KK179 sample did produce a positive
hybridi
s
ation signal, albeit at a much reduced level. These data show a clear reduction
in the level of
CCOMT
m
R
NA

in KK179 compared to the conventional control and hence
that insertion of the
CCOMT

suppression cassette has resulted in the intended
modification.


3.6

Stability of the genetic change



The concept of stability encompasses both the genetic and phenotypic
stability of the
introduced trait over a number of generations. Genetic stability refers to maintenance of the
modification over successive generations, as produced in the initial transformation event. It
is best assessed by molecular techniques, such as S
outhern analysis or PCR, using probes
and primers that cover the entire insert and flanking regions. Phenotypic stability refers to
the expressed trait remaining unchanged over successive generations. It is often quantified
by a trait inheritance analysis
to determine Mendelian heritability via assay techniques
(chemical, molecular, visual).


Genetic stability was assessed by Southern blot analyses of genomic DNA isolated from
verified leaf tissue from P
0
, MBC1, MBC2 and Syn1 generations (refer
to
Figure 3
)

of

KK179
and from C
0
. Two ra
d
iolabelled probes covering the T
-
DNA I in plasmid PV
-
MSPQ12633
were utilized following digestion with two restriction enzymes. Any instability associated with
the insert would be detected as novel bands on the Southern blot. T
he analyses showed that
the hybridization bands specific to the insert were identical for DNA from the P
0
, MBC1,
MBC2 and Syn1 generations of KK179, and hence that the insert is stably inherited from one
generation to the next. No hybridization bands were
obtained for DNA from C
0
.


Mendelian inheritance

was assessed using

leaf tissue from

verified

plants of
the
MBC2,
MBC3 and Syn1
generations (refer
to
Figure 3
)
.
Genomic

DNA was i
solated from leaf discs
and
endpoint TaqMan
PCR analysis was done using
primers

and probes

specific

to the

KK179 insert
.
A chi
-
square

(
Χ
2
)

analysis of the segregation data

over each of the
generations was used to test t
he hypothesis that the insert was

inherited according to
Mendelian princip
les i.e the

segregation

was appr
oxi
mately 1:1 (presence:absence) in the
MBC2 and MBC3 generations and approximately 3:1 in the Syn1 generation.

The
Χ
2
values
16

obtained confirmed that the hypothesis was correct and
also
suppor
ted the conclusion that
the KK179

insert has been stably integrated

into a single locus in the KK179

genome.


Phenotypic stability was also
indirectly
assessed
by measuring the total lignin content

(Acid
Detergent Lignin = ADL)

of the forage of KK179 over three generations (
MBC1, Syn1 and
Syn1
-
Adv
).

The forage was grown on replicated plots at two sites in the U.S. and was
analysed for total lignin (acid detergent ligni
n). Non
-
GM controls
(
of each generation
)

were
also grown and analysed. For each generation, the mean %dw of lignin
in the KK179 forage
was significantly (P
<0.05) less than in the control

for
age (Table 4).


Table
4
: Levels of acid d
eter
gent lignin in various generations of KK179 and non
-
GM control
plants

Generation

ADL in
Control (A)

(%dw)

ADL in

KK179 (B)

(%dw)

A vs B
(P
-
value)

MBC1

5.31 ± 0.36

4.37 ± 0.36

0.002

Syn1

4.64 ± 0.36

4.02 ± 0.36

0.034

Syn1
-
Adv

4.62 ± 0.36

3.91 ± 0.36

0.016


3.7

Antibiotic resistance marker genes


No antibiotic marker ge
nes are present in lucerne KK179
. Plasmid backbone analysis
and T
-
DNA II analysis
(refer
to
Section 3.4.1
)

show

that



no plasmid backbone has
been integrated into the lucerne

genome during
transformation, i.e. the
aadA

gene, which was used as a bacterial selectable marker
gene,

is not pr
esent in lucerne KK179



the
nptII

gene
.
incorporated in T
-
DNA II during the initial transformation procedure
has been segregated out of KK179 by traditional breeding.


3.8

Conclusion


Comprehensive molecula
r anal
yses of lucerne line KK179

indicate

there is a single insertion
site
containing

a single copy
of the T
-
DNA

I

(containing the
CCOMT

suppression cassette)
from
plasmid
PV
-
MSPQ12633
.

No DNA sequences from
T
-
DNA II or from the
backbone of
the transformation vector, including antibiotic resistanc
e marker

gene
s, were transferred

to
the plant
.
As expected from the nature of the genetic modification, there has been
a
reduction in the mRNA produced from the endogenous
CCOMT

gene.
The introduced
genetic elem
e
nts are stably inherited fr
o
m one generation

to the next.


4.

C
haracterisation of novel
substances


T
he
CCOMT

partial sequence
introduced into line KK179 is
derived from an endogenous
gene already present in lucerne and therefore its presence in the plant is not novel. It is
designed to give rise to a

non
-
coding
dsRNA
. Translation
of this dsRNA is considered
unlikely

because the
hairpin
secondary structure prevents engagement of the 40S ribosomal
subunit necessary to initiate translation at the 5’ end of the RNA, and/or it prevents
unwinding of the dupl
ex such that the 40S subunit is unable to advance along it
(Kozak,
1989)
.

As
discussed in Section 3.2.1, such

dsRNA is also cleaved into
small ds
RNAs which
themselves would have limited potential for translation.

Therefore, no novel proteins are
produced as a consequence of the genetic modification.

17

Any

small dsRNAs
produced
in line KK179

do not present a safety concern.

Small RNAs
in
general
are abundantly present in the human diet from both plant and animal sources
(Ivashuta
et al
., 2009; Carthew and Sontheimer, 2009)
,

and small RNAs have been identified
that are associated specifically with the endogenous regulation of lignin biosynthesis
(see
e.g. Ong and Wickneswari, 2012)
.



Given the absence of any novel protein, and the lack of safety concerns associated with the
production of

small dsRNAs, t
he
remainder of the characterisation

focusses

on the potential
toxicity/allergenicity of any new ORFs created by the insertion of new genetic material into
the plant genome, which may potentially
or theoretically
give rise to novel proteins
.


4.1

Potential allergenicity/toxicity of
any
novel
ORFs created by the
transformation procedure


Studies

submitted:


2012. Bioinformatics Evaluation of the Transfer DNA Insert in KK179 Utilizing the AD_2012,
TOX_2012 and PRT_2012 Databases.
MSL0024048
.
Monsanto Company (unpublished)

2012. Bioinformatics Evaluation of DNA Sequences Flanking the 5' and 3' Junctions of Inserted DNA
in KK179: Assessment of Putative Polypeptides.
MSL0023975
. Monsanto Company (unpublished)


As described
in Section 3.4.2,
translated

sequences in the insert and flanking regions were
obtained. In the case of the insert, all possible sequences in the si
x reading frames were
used in the bioinformatic analysis
, while for the flanking regions,
ten

sequences of 8 amino
acids or gr
eater (i.e.
ORFs
) were identified for bioinformatic analysis.


Assessment of potential allergenicity


To evaluate the similarity to known allergens of proteins that might potentially be produ
ced
f
rom translation of the
sequences
, an epitope search was carr
ied out to identify any short
sequences of amino acids that might represent an isolated shared allergenic epitope. This
search compared the sequences with
1,603 sequences

in the Allergen, Gliadin and Glutenin
sequence database

(designated AD_2012)
, residin
g in the FARRP (Food Allergy Research
and Resource Program) dataset (Version 10) within AllergenOnline (University of Nebraska;
http:www.allergenonline.org/
)
. The FASTA algorithm
(Pearson and Lipman, 1988)
, version
3.4t 26

(July 7, 2006)
was used to search the database using the BLOSUM50 scoring matrix
(Henikoff and Henikoff, 1992)
.

The BLOSUM series of matrices tabulate the frequency with
which different substitutions
occur in conserved blocks of protein sequences and are
effective in identifying distant relationships.
In addition, each sequence was used as a query
for an eight amino acid sliding window search

(Metcalfe
et al
., 1996)

of the AD_2012
database.



For the insert, no alignments with any of the
query
sequences generated an E
-
score
3

of


≤1e
-
5
,
and
no alignment met or exceeded the Codex Alimentarius
(Codex, 2003)

FASTA
alignment threshold for potential allergenicity
. A single potential immunologi
cally relevant
sequence of eight contiguous amino acid
s

was detected

in an eight amino acid sliding
window search.
The alignment was to a region in whe
at (
Triticum aestivum
) dehydrin and
was
shown, in KK179, to be due to a sequence in
the
CCOMT

fragment
. As discussed in the
introductory paragraph
to Section 4
, the RNA

transcribed from the
CCOMT

fragment will fold




3

Comparisons between highly homologous proteins yield E
-
values approaching zero, indicating the very low
probability that such matches would occur by chance. A larger E
-
value indicates a lower degree

of similarity.
For
FASTA searches,

hits with E
-
values of 10
-
6

or less
imply homology but
any conclusions reached need to be
tempered by an investigation of the biology behind the putative homology
(Baxevanis, 2
005)
. In this application
an E
-
value of 10
-
5

or less was set as the high cut
-
off value for alignment

significance.

18

back on itself to form a hairpin
structure

which is subsequently further processed
, making
translation unlikely
.

Furthermore, the CCOMT fragment is derived from a gene that is
already naturally present and expressed in lucerne. The identified sequence similarity
with a
wheat sequence
is
there
fore of no
sign
i
ficance
.


For the 10 ORFs in the flanking regions,
no alignments with any of the query sequences
generated an E
-
score of ≤1e
-
5
,

no alignment met or exceeded the Codex Alimentarius
(Codex, 2003)

FASTA alignment threshold for potential allergenicity
, and no alignments of
eight or more consecuti
ve identical amino acids were f
oun
d between any query sequence
and the

sequences in the

AAD_2012 database.


Assessment of potential toxicity


The
KK179
sequences were also compared with
24,731,719

sequences present in the
GenBank
protein
database (
http://www.ncbi.nlm.nih.gov/genbank/
)
, release 187.0
(designated PRT
_2012)
, which contains 12,866 toxin proteins,

using the FASTA algorithm.
No significant similarities of
either
the KK179 insert sequences

or flanking region ORF
sequences

to any
toxin sequences in the database
were found.


Conclusion


It is concluded that, in the unlikely event transcr
iption and translation of

frames 1


6 of the
inserted T
-
DNA I sequences or

flanking region
ORFs could occur, the encoded polypeptides
do not share any significant similarity with known allergens or toxins.


5.

Compositional analysis


The main purpose of compositional analysis is to determine if any unexpected changes in
composition have occurred
to the food and to establish its nutritional adequacy
.
Compositional analysis can also be important for evaluating the intended effect where there
has been a deliberate change to the composition of food.


The classic approach to the compositional analysis
of GM food is a targeted one
;

r
a
ther than
analysing every single constitu
ent, which would be impractical
, t
he aim is to analyse only
those constituents most relevant to the safety of the food or that may have an impact on the
whole diet
.
Important analytes

therefore include the key nutrients, toxicants and anti
-
nutrients for the food in question
.
The key nutrients and anti
-
nutrients are those components
in a particular food that may have a substantial impact in the overall diet
.
They may be major
constituen
ts (fats, proteins, carbohydrates or enzyme inhibitors as anti
-
nutrients) or minor
constituents (minerals, vitamins)
.
Key toxicants are those toxicologically significant
compounds known to be inherently present in an organism, such as compounds whose toxic

potency and level m
ay be significant to health (eg
solanine in potatoes)
.


5.1

Key components

of
lucerne


Main indicators of alfalfa quality for livestock feeding
(OECD, 2005)

include the proximates,
acid detergent fibre, neutral detergent fibre, lignin, and minerals and also saponins,
condensed tannins, oestrogen agonists and antagonists and cyanogenic glycosides. OECD
(2005)

suggest
s

that a minimum compositional analysis where alfalfa is likely to be sold for
food use

would be the analysis of fresh forag
e or sprouted alfalfa seed for
crude protein, fat,
ash,
fibre,
lignin, amino acids, minerals

with the addition of
vitamin C, beta
-
carotene, folate
and phytoestrogens to
provide a basis for assessment of potential uninte
nded effects with
relevance to human
food use.

Analyses for key components wer
e done on
fresh
forage (see
Section 5.2).

19


Studies

submitted:



2012. Composition Analyses of Forage from KK179 Alfalfa Grown
in the United States during the
2011 Growing Season.

MSL0023847
. Monsanto Company (unpublished)

2012. Amended Report for
MSL0023982: Composition of Lignin of Forage from KK179 Alfalfa Grown
in

the United States during the 2011 Growing Season.
MSL0024403
. Monsanto

Company
(unpublished)
.

2012. Analyses of Lignin
in Forage from KK179 Alfalfa Grown in the United States during the 2011

Growing Season.
MSL0024120
. Monsanto Company (unpublished)

2012. Analyses of Saponin Levels

of
Forage from KK179 Alfalfa Grown in the United States durin
g
the
2011
Growing Season.
MSL0023980
. Mo
nsanto Company (unpublished)


5.2

Study design
, conduct

and
analysis


The test (PCR
-
verified KK179
, s
eed of
Syn1 generation), and control (PCR
-
verified C
0

Syn1
)
lines were grow
n u
nder
agronomic conditions pertinent to their
geographic regions

at six

field sites across North America
4

during th
e 2011

growing season.
The sites were

representative of where lucerne

is commercially grown.
Four
teen

differen
t non
-
GM lucerne

lines

(PCR
-
verified)

were also grown under the same conditions
in order to generate a
reference

range for each analyte.

All lines
were planted in a randomised c
omplete block
design, with four

rep
licated plots at each of the
six

sites.


F
orage from
the lines was harvested
(first cut)
approximately 6 cm above the soil surface
when the plants were
between 1% and 10% bloom

(Ball, 1998)
, a stage between growth
stages 4 and 5 as described by Mueller & Teuber
(2007)

and recognised as the ideal stage
at which to harvest forage for maximum yield and minimal loss

of quality.
It was immediately
frozen

and then shipped to a laboratory for grinding to powder before frozen storage.
Methods of composition analysis were based on internationally recognised proc
edures (e.g.
thos
e of the Association of Analytical Communities
-

AOAC
),

methods specified by the
manufacturer of the equipment used for analysis, or other published methods.


For each analyte ‘descriptive statistics’ were generated i.e. a mean (least square mean) and
standard error
(S.E.)
averaged over all sites (combined
-
site

analysis). The values thus
calculated

are presented
in
Tables 5


9
.



The analytes were analysed usin
g a mixed model analysis of variance. Data were
transformed into Statistical Analysis Software
5

(SAS) data sets and analysed using SAS®
software (SAS MIXED). The four replicated sites were analysed both separately and
combined across all sites (combined
-
si
te analysis). In assessing the significance of any
difference between means, a P
-
value of 0.05 was used (i.e. a P
-
value of ≥0.05 was not
significant).


Any statistically significant differences between
KK179 and
the
C
0

Syn1

control have been
compared to the 95% tolerance interval (i.e. 95% confidence that the interval contains 99%
of the values expressed in the commercial lines) compiled from the results of the
fourteen

commercial reference lines combined across all sites, to

assess whether the differences are
likely to be biologically meaningful. Additionally,
the results for
KK179 and the
C
0

Syn1

have
been compared to
a

combined literature range for each analyte, compiled from published




4

The six

sites were:
Tulare County, CA; Jefferson County, IA; Clinton County, IL; Pawnee County,
KS, Armstrong County, TX and Walworth
County, WI.

5

SAS website
-

http://www.sas.com/technologies/analytics/statistics/stat/index.html

20

lit
erature
6
.
Any mean

value for a lucerne KK179

analyte that fell within the combined
literature range was considered to be within the normal v
ariability of commercial lucerne

cultivars even if the mean value was statistica
lly different from the C
0

Syn1
control. It is
noted, h
owever, that information in the published literature is limited and is unlikely to
provide
a broad reflection of the natural diversity that occurs within
lucerne
. Therefore, even
if means fall outside the published range, this is not necessarily a concern.


5.3

Forage

composition


Forage samples were analys
ed for nutrients; proximates (ash, fat, moisture, and protein),
carbohydrates by c
alculation, acid detergent fibre (ADF), neutral detergent fibre

(NDF), acid
detergent lignin (ADL), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, and Zn), and amino acids
(essential and non
-
essential). Antinutrient and secondary metabolites included daidzein,
glycitein, genistein, coumesterol, formononetin, biochanin A, and

canavanine. In addition to
the OECD
(OECD, 2005)

recommended analytes,
p
-
coumaric acid, ferulic acid, sinapic acid,
total polyphenols and fre
e phenylalanine were also analys
ed to evaluate the
potential
effect
of
CCOMT

suppression on lignin pathway and cell wall
-
associated metabolites.


5.
3.1

Proximates and fibre


Results of the proximate and fibre analysis are shown
in
Table 5
.

With regard to lignin level,
two separate analyses

using two different methods
(designated ADL1 and ADL2 in
Table 5
)

were done on plants from the same trial.
The major differences between the analyses were
in sample fineness of grind and method au
tomation.

It is noted that in the ADL1 lignin, there
is no
statistically
significant difference between KK179 and the control (although the actual
numerical value is lower in KK179), while in the ADL2 lignin, the level is
statistically
significantly lower
in KK179 compared to the control.

It is accepted, considering also

the
results provided both in Section 3.6 (Table 4) and in
Table 6 that there has been a genuine
reduction in th
e level of lignin in line KK179 compared with the control,

even though the
red
uced level
is higher than the
level at the
lower end of
both the reference range and the
combined literature range.

It is noted that

there would be a limit to the amount of reduction in
lignin that would be agronomically acceptable

since too big a reduction

would

result in

the
lodging of plants. It is further noted that the predominant purpose of the genetic modification
in KK179 is to provide an agronomic
benefit
r
ather than a nutritional change

per se
.
It has
long been understood
that dietary lignin is not appreciably metabolized by animals
(Crampton and Maynard, 1938)
. The amount deposited in cell walls increases with
increasing plant maturity thereby reducing forage quality. Therefore when a crop is used for
forage there will be a trade
-
off between maximising yield and minimising lignin content.
For
line KK179, a redu
ction in lignin means that
plants could be harvested later

than standard
lines to get the same forage quality but higher yield.


The only
other
significant difference occurred in a comparison of the
ash in C
0

Syn1 and
KK179 where the latter had a lower

me
an value. However, this mean was well within
both
the reference range and the range reported in the literature.









6

References included:
Smith
(1969)
; Jung & Fahey Jr.
(1983)
;
Natelson & Bratton
(1984)
;
Cherney
et

al.
(1989)
;

Bourquin

et al.
(2013)
; Rosenthal & Nkomo
(2000)
;
OECD
(2005)
;
McCann et al
(2013)
; Dairyland Laboratories
( 2012)
.

21

Table
5
: Mean percentage

± S.E.

of
proximates

and fibre in forage from C
0

Syn1 and KK179

Analyte

‘C
0

Syn’ (A)

KK179
(B)

A vs B
(P
-
value)

Reference
range

Combined
literature

range

Protein

(%
dw
)

21.02 ± 1.35

20.83 ± 1.36

NS

14.52


30.07

14.91


28.34

Fat

(%dw)

2.28 ± 0.17

2.28 ± 0.17

NS

0.53


4.21

1.3


3.24

Ash

(%dw)

10.79 ± 0.52

10.38 ± 0.53
5

0.034

7.54


13.23

5.8


15.3

Moisture

(%fw)

78.15 ±1.54

78.26 ± 1.54

NS

66.10


85.30

7.74


83.5

Carbohydrate

(%dw)
1

65.97 ± 1.70

66.55 ± 1.71

NS

54.35


74.91

56.63


74.8

ADF
2

(%dw)

27.02 ± 2.44

27.03 ± 2.45

NS

7.07


39.11

21.26


42.59

NDF
3

(%dw)

34.46 ± 2.63

33.95 ± 2.64

NS

18.97


49.82

26.5


53.56

ADL
1
6

(%dw)

6
.54 ±

0.59

6.22 ± 0.60

NS

3.38


9.67

2.31


13.71

ADL
2

(%dw)

6.93 ± 0.64

5.39 ± 0.64

0.004

1.70


10.03

2.31


13.71

1
Carbohydrate calculated as 100%
-

(protein %dw + fat %dw + ash %dw)

2
ADF =
acid detergent fibre

3
NDF = neutral detergent fibre

4
ADL = acid detergent lignin

5
Mauve shading

represents
a
KK179 mean

with
a

significantly lower
value than

the C
0

Syn1

mean
.

6
See text above for explanation of ADL1 and ADL2


Lignin components


Section
3.2.1

describes

the
three main components that go to make up lignin in lucerne.


A
n

analysis of the
se

individual components of lignin in KK179 and the
C
0

Syn1 control was
done using data collected at the same time from the same sites and with the same 14
r
eference varieties.
Forage samples were analysed for levels of p
-
hydroxyphenyl (H) lignin,
caffeyl (C) lignin, guaiacyl (G) lignin, 5
-
hydroxyguaiacyl lignin, and syringyl (S) lignin units.
The C lignin and 5
-
hydroxyguaiacyl lignin unit components were belo
w the limit of
quantitation for all samples and were excluded from the statistical analysis. The results for
the remaining lignin components are given in
Table 6
.

These

results

considered
the analytes
in terms of a) the amount of each analyte present per cell wall residue (CWR); b) the
proportion of each analyte in the total
(
H + G + S
)

lignin
; and c) the ratio of S:G

lignin.

As

expected from the intention of the genetic modification,

the m
ean level of G lignin in
KK179 was significantly lower than the mean in
C
0

Syn1

and the proportion of G lignin
expressed in the total (H + S + G) lignin was significantly lower in KK179 compared to
C
0

Syn1
. To counteract this lower proportion of G lignin i
n the total, there was a corresponding
increase in the proportions of H and S lignin and in the S:G ratio in KK179.


The results support the conclusion that suppression of the endogenous
CCOMT

gene
, as a
result of the genetic modification, acts to decrease

the amount of G lignin.




22

Table
6
: Summary of
forage lignin unit content in C
0

Syn1 and KK179

Lignin
Component

‘C
0

Syn’ (A)

KK179 (B)

A vs B
(P
-
value)

Reference
range

Combined
literature

range

µmole/g CWR

Guaiacyl lignin

83.72 ± 9.40

68.10 ± 9.48

0.027

25.34


153.11

NA

Hydroxyphenyl
lignin

3.88 ± 0.43

5.05 ± 0.45

NS

0.29


8.26

NA

Syringyl lignin

50.41 ± 8.78

55.96

±
8.83
5

NS

5.64


110.93

NA

% Total (H + G + S)

Guaiacyl lignin

61.69

±
1.87

53.69

± 1.
87

<0.001

50.02


76.69

NA

Hydroxyphenyl
lignin

3.07

±
0.54

4.22

±
0.54

0.001

0.18


6.23

NA

Syringyl lignin

35.24

± 2.
35

42.09

± 2.
35

<0.001

17.07


46.14

NA

Ratio

S:G

0.58

±
0.060

0.80

±
0.060

<0.001

0.22


0.92

NA


5.3.2

Amino acids


Levels of 18 amino acids were
measured. Since asparagine and glutamine are converted to
aspartate and glutamate respectively during the analysis, levels for aspartate include both
aspartate and asparagine, while glutamate levels include both glutamate and glutamine.


Results of the ana
lysis are given
in
Table

7
.

There was no significant difference between
C
0

Syn1 and KK179
f
or any of the amino acids and all means fell within both the reference
range and literature range.


Table
7
: Mean percentage dry weight (dw
)
± S.E.
, relative to total dry weight, of amino acids
in forage from C
0

Syn1 and KK179

Amino Acid

‘C
0

Syn’ (A)
%dw

KK179 (B)
%dw
1

A vs B

(P
-
value)

Reference
range

%dw

Combined
literature

range

%dw

Alanine

1.13 ± 0.074

1.11 ± 0.074

NS

0.80


1.66

0.70


1.59

Arginine

1.01 ± 0.065

0.99 ± 0.065

NS

0.70


1.44

0.62


1.54

Aspartate

2.74 ± 0.28

2.77 ± 0.28

NS

1.96


5.15

1.40


3.52

Cysteine

0.21 ± 0.011

0
21 ± 0.012

NS

0.16


0.31

0.18


0.35

Glutamate

1.91 ± 0.12

1.85 ± 0.12

NS

1.31


2.80

1.20


3.03

Glycine

0.97 ± 0.055

0.95 ± 0.055

NS

0.70


1.33

0.60


1.47

Histidine

0.44 ± 0.020

0.43 ± 0.020

NS

0.34


0.61

0.28


0.74

Isoleucine

0.88 ± 0.053

0.86 ± 0.053

NS

0.63


1.27

0.50


1.26

Leucine

1.47 ± 0.089

1.43 ± 0.089

NS

1.03


2.05

0.90


2.25

Lysine

1.17 ± 0.067

1.14 ± 0.067

NS

0.82


1.73

0.59


1.81

23

Amino Acid

‘C
0

Syn’ (A)
%dw

KK179 (B)
%dw
1

A vs B

(P
-
value)

Reference
range

%dw

Combined
literature

range

%dw

Methionine

0
.24 ± 0.024

0.
25 ± 0.024

NS

0.
14


0.45

0.18


0.48

Phenylalanine

1.00 ± 0.061

0.98 ± 0.061

NS

0.71


1.39

0.72


1.59

Proline

0.92 ± 0.053

0.89 ± 0.054

NS

0.65


1.25

0.70


1.34

Serine

0.88 ± 0.044

0.87 ± 0.044

NS

0.66


1.25

0.60


1.36

Threonine

0.88 ± 0.050

0.86 ± 0.050

NS

0.63


1.23

0.60


1.15

Tryptophan

0.
37 ± 0.020

0
.37 ± 0.020

NS

0.
25


0.50

0.16


0.35

Tyrosine

0.71 ± 0.042

0.71 ± 0.042

NS

0.52


1.01

0.50


1.16

Valine

1.07 ± 0.061

1.
05 ± 0.061

NS

0.79


1.55

0.60


1.55


5.3.3

Minerals


Levels of 9 minerals were measured. The means for these are given
in
Table

8

and

show
there was no significant difference between
C
0

Syn1 and KK179
for any of the minerals. All
means fell within both the reference range and literature range.


Table
8
: Mean values ± S.E. for mineral levels in forage from C0 Syn1 and KK179

Mineral

‘C
0

Syn’ (A)

KK179 (B)

A vs B
(P
-
value)

Reference
range

Combined
literature

range

Calcium
(%dw)

1.72 ± 0.16

1.68 ± 0.16

NS

0.95


2.07

0.90


1.96

Copper
(mg/kg dw)

8.34
±

0.85

8.86
±

0.85

NS

4.54


19.67

3.43


14.72

Iron

(mg/kg dw)

315.74 ± 30.93

272.00 ± 31.45

NS

105.45


691.43

0.2
-

4749

Magnesium

(%dw)

0.23 ±

0.023

0.22 ± 0.023

NS

0.11


0.34

0.11


0.45

Manganese
(mg/kg dw)

52.45 ± 6.27

52.56 ± 6.30

NS

23.24


98.04

15.91


109.5

Phosphorus

(%dw)

0.28 ± 0.019

0.29 ± 0.019

NS

0.18


0.43

0.22


0.46

Potassium
(%dw)

2.41 ± 0.051

2.35 ± 0.052

NS

1.85


3.35

1.39


4.31

Sodium

(%dw)

0.077 ± 0.024

0.089 ± 0.024

NS

0.016


0.20

0.017


0.51

Zinc

(mg/kg dw)

26.81 ± 2.09

27.83 ± 2.11

NS

17.08


47.48

15.2


43.62



5.3.
4

Phyto
estrogens


Phytoestrogens are naturally
-
occurring plant compounds that are structurally and/or
functionally similar to mammalian
o
estrogens and their active metabolites. Most are phenolic
compounds of which the isoflavones

and coumestans are the most widely researche
d
groups
(Patisaul and Jefferson, 2010)
. They are
ubiquitous in the plant kingdom but are
found particularly in soy and other legumes
(Kurzer and Xu, 1997; Setchell, 1998)
.


24

Levels
of
daidzein, glycitein, genistein, formononetin and biochanin A (isoflavones), and
coumestrol (coumestan) were measured
in
forage from
KK179
,

C
0

Syn1
and the fourteen
reference varieties. For all phytoestrogens except coumestrol, all levels across a
ll sites in all
lines were below
the

L
imit of Qu
an
titation
(LOQ).

For

coumestrol approximately 80% of
levels across all sites and all lines were below the LOQ.

Statistical analysis of the
phytoestrogens was therefore not meaningful.


5
.3
.
5

Other analytes


A number of other analytes were included in the compositional analyses.
The OECD
(OECD,
2005)

suggest
s

that
saponins and
canavanine
also
be considered in a
forage
compositional
analysis.
Saponins are found in many

plants, including edible species,
but particularly
legumes
(Deshpande, 2002)
.


In animal forage they have implications for bloating
(OECD,
2005)
. In the hum
an diet, their

significance in either a beneficial or adverse role is unclear
(Deshpande, 2002)
.
Saponin analysis included measurement of levels
of total saponins, and
individual saponin compounds, specifically, bayogenin, hederagenin, medicagenic acid,
soyasapogenol B, soyasapogenol E, and zanhic acid.



Canavanine is a potentially toxic structural analogue of L
-
arginine that is a stored by
many
l
egumes including alfalfa
(Rosenthal and Nkomo, 2000)
.


Perturbations in the lignin biosynthetic
pathway
(see Figure 2)
may produce effects that go
beyond alterations in lignin amount, composition, and cell wall structure and have the
potential to affect the expression level of other lignin pathway genes
(Vanholme
et al
., 2010)
.
Analyses were
therefore also
done of the following p
oten
tially affected analytes:

p
-
coumaric
acid, ferulic acid, sinapic acid, total polyphenols and free phenylalanine.


Too many measurements of sinapic acid were below the
LOQ to allow a statistical analysis
of this analyte to be carried out.
The result
s for the remaining analytes

are given in
Table 9
.



There were no significant differences between KK179 means and the control means for any
of the saponins. The mean canavanine level was significantly lower in KK179 than in
C
0

Syn1
but
was within the reference range. B
oth the KK179 and C
0

Syn1
means were be
low

the literature range;

i
t is noted, howeve
r, that t
his range was compiled from
only one
reference

(Natelson and Bratton, 1984)
.

It is further noted

that the leaves of alfalfa forage
contain much lower levels of canavanine than seeds and sprouts. Typical levels of
canavanine in sprouts is around 20,000 ppm
(Rosenthal and Nkomo, 2000)
.


The ferulic acid mean was significantly higher in KK179 compared to the control but was still
within both the reference range and literature range


Table
9
:
Mean levels ± S.E.
of other
analytes considered in forage from C0 Syn1 and KK179

Analyte

‘C
0

Syn’ (A)

KK179 (B)

A vs B
(P
-
value)

Reference range

Combined
literature

range

Total bayogenin
(response units /µg)

5.67 ± 0.76

5.10 ± 0.76

NS

1.46


11.28

NA

Total hederagenin

(response units /µg)

3.47 ± 0.35

2.94 ± 0.35

NS

0.90


10.31

NA

Total medicagenic acid

(response units /µg)

23.39 ± 2.44

21.88 ± 2.44

NS

2.04


48.33

NA

Total soyasapogenol B

(response units /µg)

24.53 ± 3.02

22.17 ± 3.02

NS

9.22


43.87

NA

Total
soyasapogenol E
(response units /µg)

3.08 ± 0.54

2.77 ± 0.54

NS

0.91


7.53

NA

25

Analyte

‘C
0

Syn’ (A)

KK179 (B)

A vs B
(P
-
value)

Reference range

Combined
literature

range

Total Zanhic acid

(response units /µg)

5.16 ± 0.58

4.59 ± 0.58

NS

1.75


13.20

NA

Total saponins

(response units /µg)

65.58 ± 4.94

59.30 ± 4.94

NS

17.38


103.19

NA

Canavanine

(ppm dw)

57.24 ± 13.51

40.30 ± 13.53
1

0.013

11.47


151.33

6
00


1,
200

Ferulic acid

(ppm dw)

1485.81 ± 58.83

1596.41 ± 59.57
1

0.008

1103.32


1906.86

627
-

2840

Free phenylalanine

(ppm dw)

283.70 ± 28.69

266.99 ± 28.84

NS

133.05


579.05

NA

Total polyphenols

(mg/g dw)

7.99 ± 0.34

8.19 ± 0.34

NS

6.17


11.17

NA

p
-
Coumaric acid

(ppm dw)

623.54 ± 37.34

639.50 ± 37.62

NS

326.19


945.58

398
-

1860

1

Mauve shading represents

a

KK179 mean
that was

significantly lower
than

the C
0

Syn1 mean; orange shading
represents a significantly higher mean value for KK179 compared with C
0

Syn1.


5.3.6

Summary of analysis of key components


A total of 50

analytes were analysed.
In addition to
the intended difference in lignin
generally
,

and G lignin in particular, s
tatistically significant differences in the
three
analyte
level
s found in forage of lucerne KK179 and
C
0

Syn1

are summarised
in

Table 10
.
:



Table
10
: Summary of analyte means found in forage from KK179 that are significantly
( P<0.05) different from those found
in forage of the control line C
0

Syn1

Analyte

‘C
0

Syn’

KK179
1

%
difference

KK179
within
Reference
range?

KK179
within
Combined
literature

range?

Ash

(%dw)

10.79

10.38
1

3
%

yes

yes

ADL 2

(%dw)

6.93

5
.39

23%

yes

yes

Guaiacyl lignin
(µmole/g CWR)

83.72

68.10

19%

yes

NA

Guaiacyl lignin
(% Total
H+G+S)

61.69

53.69


13%

yes

NA

Canavanine

(ppm dw)

57.24

40.30
1

29%

yes

No


but
neither was
the control

Ferulic acid

(ppm dw)

1485

1596
1

7%

yes

yes

1

Mauve shading represents a KK179 mean that was significantly lower than the C
0

Syn1 mean; orange shading
represents a significantly higher mean value for KK179 compared with C
0

Syn1.


5.4

Conclusion


As expected, the level of lignin in general, and G lignin in particular, in KK179 were
statistically
significantly lower than in the control. However the levels were within
the
reference range obtained for non
-
GM reference varieties grown at t
he same time
.
While the
difference in lignin levels between the GM line and the control is of agronomic significance,
in that it enables the forage to be harvested at a later date without appreciable loss of forage
quality, it is unlikely to have any nutri
tional significance to humans given the range of natural
variation that exists in lucerne

26

For the remaining analytes,
statistically significant differences were noted in only three
analytes (
ash, canavanine and ferulic acid
). In all cases the differences
were typically small
and within the reference range obtained for non
-
GM reference varieties grown at the same
time. Any observed differences are therefore considered to represent the natural variability
that exists within lucerne.



7
.

Nutritional impact



In assessing the safety of a GM food, a key factor is the need to establish that the food is
nutritionally
adequate and will
support typical growth and well
-
being
.
In most cases, this can
be achieved through an understanding of the genetic modification an
d its consequences,
together with an extensive compositional analysis of the food.


I
f the compositional analysis indicates biologically significant changes to the levels of certain
nutrients in the GM food, addition
al nutritional assessment

should
be
undertaken to assess
the consequences of the changes and determine whether nutrient intakes are likely to be
altered by the introduction of such foods into the food supply
.


Where a GM food has been shown to be compositionally equivalent to conventional
v
arieties,
as is

the case for lucerne line KK179
,
the evidence to date indicates that feeding
studies using target
livestock species will add little
value
to the safety assessment and
generally are not warranted
(OECD, 2003; EFSA, 2008)
.
Lucerne KK179 is the result of a

genetic modification to
silence the expres
sion of an endogenous gene,
with

the intention of
altering an agronomic characteristic. T
he extensiv
e compositional analyses of forage,

that
have been undertaken to demonstrate the nu
tr
itional adequacy of line KK179
, indicate it is
equivalent in com
positio
n to conventional lucerne

cultivars.



The Applicant did, however supply a lamb feeding study which has been evaluated by
FSANZ.


Study

submitted:



2012.
Alfalfa hay from KK179 is wholesome when fed to growing lambs.
MSL0023898
.
Monsanto
Company

(unpublished)
.


The analysis did not show any significant difference in the measured parameters (which
included growth performance, blood chemistries and necropsy data) and general health
between lambs fed a diet containing KK179 hay and those fed a diet
containing hay from a
conventional control (C
0
-
Syn Adv). This was consistent with the findings from the
compositional analysis.


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